The invention relates to a flow through cartridge for selecting an analyte online with high performance liquid chromatography.
One example for selecting an analyte for detection using high performance liquid chromatography (also known as high pressure liquid chromatography) is solid phase extraction (SPE). SPE uses the affinity of a substance or substances in a liquid (the mobile phase) for a solid (the stationary phase) along which the liquid is passed to separate the substance or substances from the remainder of the sample. The result is that either the substance or substances separated out are retained on the stationary phase. The portion that passes through the stationary phase is collected or discarded, depending on whether it contains the desired analytes or an undesired remainder. If the desired analytes have been retained on the stationary phase, they can then be removed from the stationary phase for collection in an additional step, in which the stationary phase is rinsed with an appropriate eluent, optionally after rinsing to remove substances that have been retained but are not part of the analyte that is subject of further analysis. Conversely, the liquid that has passed along the solid can be analyzed in a next step.
SPE may for instance be carried out as follows:
a) conditioning a sorbent in a cartridge, a liquid suitable for conditioning being passed through the cartridge;
b) applying a sample that contains the analyte to the sorbent, a mobile phase liquid which contains the sample being passed through the cartridge;
c) washing the sorbent, a wash liquid being passed through the cartridge; and
d) eluting the analyte from the sorbent, an elution liquid being passed through the cartridge.
Step a) serves to wet the surface of the sorbent to create the stationary phase that absorbs the analyte. In step b) the substance to be tested, the analyte, is applied to the sorbent and, at least to a useful extent, selectively absorbed by the sorbent. In step c) the sorbent is washed so that constituents which could interfere with detection of the analyte are removed. In step d) the analyte is eluted from the sorbent so that it can be detected in a following step, for example by gas chromatographic analysis (GC) or by means of high performance (or pressure) liquid chromatography (HPLC).
Already for quite some time, in particular for automation of bioanalytical assays, especially in the pharmaceutical industry, SPE is more and more carried out in direct communication with HPLC or Mass Spectrometry (MS) downstream of the SPE (usually referred to as “online” or “on-line” with the SPE). Total automation, high precision and high sensitivity are among the most favoured features of online SPE. An example of online SPE and HPLC is described in applicant's European patent 1 159 597 and in Recent Developments in On-line SPE for HPLC and LC-MS in Bioanalysis; Emile Koster and Bert Ooms, Guide to LC-MS, December 2001, pp 1-3.
Modern HPLC systems work at high pressures, and therefore are able to use very small particle sizes in the columns (<2 μm). It is known to apply pressures in such “Ultra High Performance (or Pressure) Liquid Chromatography” systems (UHPLCs) of up to 100 MPa.
The cartridge is typically constituted by a body through which a passage extends from one side of the cartridge to an opposite side of the cartridge, packing material and sieves. To manufacture the cartridge at low costs, the body is injection moulded, usually from Polyvinylidene Fluoride (PVDF), the sieves are welded to one side of the body, then the packing material is inserted and finally the second sieve is placed and hot welded. Polyvinylidene Fluoride is a relatively low cost fluoropolymer that is injection mouldable easily because of its low melting point (around 177° C.), quite strong and resistant to solvents acids and bases.
Prior to carrying out SPE, the cartridge is temporarily clamped between two jaws of a clamp so that passages in the jaws are connected in series with the passage through the cartridge body so as to form a continuous fluid path. Fluid can then be pumped through the cartridge to initiate interaction between components in the fluid and the packing material in the form of SPE.
A problem associated to existing SPE cartridges is insufficient resistance to pressures higher than 25 MPa. Such cartridges are unsuitable for online communication with UHPLC, because UHPLC involves the application of higher pressures (sometimes up to 120 MPa). UHPLC is becoming increasingly important for the analysis of complex samples because of its high separation power.
Another problem of existing SPE cartridges is insufficient resistance to traditional HPLC pressures at temperatures higher than around 70° C. This very much restricts the use of “Temperature Assisted Solid Phase Extraction” (TASPE), see for instance applicant's European patent 1 159 597.
More in general, the use of conventional cartridges at high pressures entails leakage problems where surfaces of the jaws and of the SPE cartridge are pressed against each other.